Mitochondria play a key role in generating ATP for energy-dependent processes in cells. Therefore, mitochondrial dysfunctions often have significant effect on cell function and survival. For example, when mitochondrial membranes are damaged, mitochondria lose their membrane potential, uncoupling oxidative phophorylation and preventing ATP production therefrom, and intra-mitochondrial solutes are released, some of which induce cell death.
One of the better-characterized mechanisms leading to pathologic increase in permeability of mitochondrial membranes is the opening of the mitochondrial permeability transition (MPT) pore. This is a high-conductance, nonspecific channel in the intra-mitochondrial membrane that conducts solutes up to 1.5 kDa. The pore opening is regulated by multiple effectors and therefore, its opening is not readily predicted.
When open, however, MPT pores lead to osmotic swelling of mitochondria, which can cause irreversible damage to their membranes. Several techniques have been proposed for assessing the induction of MPT in mitochondria by detecting the pathologic increase in their membrane permeability. These techniques are based on monitoring the translocation of a specific marker through damaged mitochondrial membranes. Thus, either mitochondria are incubated in the presence of a fluorescent or radioactive marker which is taken up only by injured mitochondria, or the mitochondria are pre-loaded with markers that are released upon a significant increase in mitochondrial membrane permeability.
These techniques, however, have several disadvantages and are not suitable, for example, in drug screening applications. Notably, both techniques require manipulations with mitochondria before the actual marker translocation is assessed. Furthermore, several control experiments are required to exclude possible respiratory inhibition, calcium uptake blocking, or uncoupling of mitochondria. Usually background signals are high, therefore significantly limiting the sensitivity of the detection assay.